The nuclear receptor superfamily constitutes a class of ligand-dependent transcriptional factors that regulate gene expression during many biological processes, including development, cellular proliferation and differentiation. The activity of these receptors is relevant to disease since alterations in receptor signaling pathways have been linked to various disease processes. RTR/GCNF is a nuclear orphan receptor that plays an important role in the control of gene expression during early embryonic development and gametogenesis. In the absence of ligand, RTR functions as a repressor of transcription. In this study, we further characterized this repressor function. We demonstrated that RTR inhibited basal transcriptional activation and suppressed the transactivation by the estrogen receptor related-receptor a1 (ERRa1) through its response element (ERRa1-RE). The latter was at least in part due to competition for binding to the same RE. Mammalian two hybrid analyses showed that the transcriptional repression by RTR can be mediated through interactions with the co-repressor N-CoR and does not involve SMRT or RIP140. Pull-down analyses with GST-RTR demonstrated the formation of RTR(:)N-CoR complexes in vitro. Deletion and point mutation analyses revealed that the hinge-domain, helix 3 and 12 of RTR are essential for its interaction with N-CoR. The residues S244Y245 in the hinge domain, K318 in helix 3 and K489T490 in helix 12 were identified as being critical for this interaction. Recently we cloned a novel gene referred to as RAP80 that is able to interact with RTR and may play a role in the repressor function of RTR. RAP80 is an 80 kD nuclear protein containing two zinc finger domains at its carboxy terminus. Although N-COR and RAP80 are able to compete for binding to RTR, different regions of RTR are involved in these interactions. Our results demonstrate that, in the absence of ligand, RTR functions as an active transcriptional repressor and that this repression can be mediated through interactions with the co-repressor N-CoR. Through its repressor function, RTR can suppress the transcriptional activation by other nuclear receptors. These repressor activities may provide important mechanisms by which RTR regulate biological processes, such as spermatogenesis.